Method for producing a modified bacteriophage without genome modification

Abstract

The invention relates to a method for producing a modified bacteriophage in a cell-free expression system wherein the expression of at least one gene of interest is suppressed by a molecule specifically inhibiting its expression. The invention further relates to a composition and a kit for producing a modified bacteriophage. Moreover, the invention relates to a bacteriophage which is not modified on the genomic level but on the proteomic level and its use for therapy, for diagnostic and detection assays.

1 . Method for producing a modified bacteriophage in a cell-free expression system comprising the steps of:
 contacting a cell lysate of a microorganism with a genome of the bacteriophage,   suppressing expression of at least one gene of interest encoded by the genome of the bacteriophage by adding a molecule specifically inhibiting the expression of the endogenous version of the at least one gene of interest.   
     
     
         2 . Method according to  claim 1 , wherein the method further comprises the step of
 expression of a modified version of the at least one gene of interest by adding a molecule encoding a modified version of the gene of interest.   
     
     
         3 . Method according to  claim 1  or  2 , wherein the method further comprises the step of
 adding a modified bacteriophage protein encoded by a modified version of the at least one gene of interest. 
 
     
     
         4 . Method according to any one of the preceding claims, wherein the gene of interest is an essential gene or a non-essential gene. 
     
     
         5 . Method according to any one of the preceding claims, wherein the genome of the bacteriophage is not modified. 
     
     
         6 . Method according to any one of the preceding claims, wherein the gene of interest encodes a capsid protein or a tail fiber protein of the bacteriophage, preferably wherein the gene of interest encodes a highly immunogeneic outer capsid protein (HOC). 
     
     
         7 . Method according to any one of the preceding claims, wherein the modified bacteriophage protein comprises a modification selected from the group consisting of an affinity tag, a detection marker, a protein for the improvement of the bacteriophage or mutation or combinations thereof 
     
     
         8 . Method according to any one of the preceding claims, wherein the bacteriophage is a bacteriophage of the Myoviridae family, preferably of the Tevenvirinae subfamily, even more preferably a T4 virus, most preferably the bacteriophage is  Escherichia  virus T4. 
     
     
         9 . Composition for producing of a bacteriophage comprising:
 a cell lysate of a microorganism,   a genome of the bacteriophage,   a molecule specifically inhibiting the expression of the endogenous version of the gene of interest,   optionally a molecule encoding a modified version of the gene of interest, and   optionally a modified bacteriophage protein encoded by a modified version of the at least one gene of interest.   
     
     
         10 . Kit for producing a bacteriophage comprising:
 a cell lysate of a microorganism,   a genome of the bacteriophage,   a molecule specifically inhibiting the expression of the endogenous version of the gene of interest,   optionally a molecule encoding a modified version of the gene of interest, and/or   optionally a modified bacteriophage protein encoded by a modified version of the at least one gene of interest.   
     
     
         11 . Bacteriophage comprising
 a genome which is not modified,   modified bacteriophage protein.   
     
     
         12 . Bacteriophage obtained by the method according to  claims 1  to  8 . 
     
     
         13 . Bacteriophage according to  claims 11  and  12  for use as a medicament. 
     
     
         14 . Bacteriophage according to  claims 11  and  12  for used in the treatment of a bacterial infection in a subject. 
     
     
         15 . Use of the bacteriophage according to  claims 11  and  12  for avoiding bacterial growth in food or beverage. 
     
     
         16 . Use of the bacteriophage of  claims 11  and  12  for detecting specific microorganisms.